Rapid detection of Norwalk-like viruses (NLVs)

Describes the development of a reverse transcription-polymerase chain reaction and an enzyme immunoassay for the detection of an Australian Norwalk-like virus group 2b virus (Camberwell) and related viruses and their application to clinical samples.Thesis (M.Sc.) -- University of Adelaide, Dept. of Microbiology and Immunology, 2000

Viperin is induced following dengue virus type-2 (DENV-2) infection and has anti-viral actions requiring the C-terminal end of viperin

The host protein viperin is an interferon stimulated gene (ISG) that is up-regulated during a number of viral infections. In this study we have shown that dengue virus type-2 (DENV-2) infection significantly induced viperin, co-incident with production of viral RNA and via a mechanism requiring retinoic acid-inducible gene I (RIG-I). Viperin did not inhibit DENV-2 entry but DENV-2 RNA and infectious virus release was inhibited in viperin expressing cells. Conversely, DENV-2 replicated to higher tires earlier in viperin shRNA expressing cells. The anti-DENV effect of viperin was mediated by residues within the C-terminal 17 amino acids of viperin and did not require the N-terminal residues, including the helix domain, leucine zipper and S-adenosylmethionine (SAM) motifs known to be involved in viperin intracellular membrane association. Viperin showed co-localisation with lipid droplet markers, and was co-localised and interacted with DENV-2 capsid (CA), NS3 and viral RNA. The ability of viperin to interact with DENV-2 NS3 was associated with its anti-viral activity, while co-localisation of viperin with lipid droplets was not. Thus, DENV-2 infection induces viperin which has anti-viral properties residing in the C-terminal region of the protein that act to restrict early DENV-2 RNA production/accumulation, potentially via interaction of viperin with DENV-2 NS3 and replication complexes. These anti-DENV-2 actions of viperin show both contrasts and similarities with other described anti-viral mechanisms of viperin action and highlight the diverse nature of this unique anti-viral host protein.Karla J. Helbig, Jillian M. Carr, Julie K. Calvert, Satiya Wati, Jennifer N. Clarke, Nicholas S. Eyre, Sumudu K. Narayana, Guillaume N. Fiches, Erin M. McCartney, Michael R. Bear

Altered responses of Dengue virus infected cells to TNF-α and induction of GRP78 and HSP70 - in vitro studies.

Dengue virus (DENV) infection of humans is characterised by immunopathology with elevated levels of many inflammatory mediators. Tumour necrosis factor alpha (TNF-α) plays a significant role in the pathogenesis of DENV infection with elevated levels of TNF-α in the sera of DENV infected patients that parallel the severity of disease and release of TNF-α coincident with the peak of DENV production from infected monocytederived-macrophages (MDM) in vitro. However, the effect of TNF-α on DENV replication is not fully clarified. In this study we aimed to determine (1) the effect of TNF-α on DENV replication and (2) the changes in host cell protein expression, in response to DENV-infection. Since macrophages are a primary cell target in vivo for DENV-infection, this study mainly used primary monocyte-derived-macrophages (MDM) and macrophagelike cell lines (K562, U937) to represent this cell type. Initially methods were developed for specific analysis of DENV replication, including a tagged RT-PCR method for quantitation of DENV positive (+ ve) and negative (- ve) strand RNA. Next the potential antiviral role of TNF-α in regulating DENV replication in MDM was investigated. While pre-treatment of MDM with TNF-α had a minor inhibitory effect, addition of TNF-α to MDM with established DENV-infection had no effect on DENV replication as measured by DENV RNA levels or virion production. Blocking endogenous TNF-α using TNF-α antibodies or TNF-α siRNA also had no effect on infectious DENV production or RNA synthesis. Together, these results demonstrate that DENV replication in MDM is not affected by TNF-α. Additionally, normal cellular TNF-α signalling, measured by quantitation of TNF-α-induced stimulation of transcription from a nuclear factor-kappa B (NF-kB) responsive reporter plasmid or NF-kB protein nuclear translocation, was blocked in DENV-infected MDM. Thus, DENV replication in MDM is not affected by TNF-α, and infected cells do not respond normally to TNF-α stimulation. It is therefore unlikely that the increased production of TNF-α seen in DENV-infection and correlating with DENV pathology contributes directly to DENV clearance by inducing anti-viral defence mechanisms and reducing DENV replication in MDM. These results also highlight an example of viral subversion of potential anti-viral cellular responses. Secondly, the host cell response to DENV-infection was analysed, presenting the first proteomic analysis on the cellular response to DENV-infection. The differential proteomes of K562 cells with or without DENV infection were resolved and quantitated with two dimensional differential gel electrophoresis (2D PAGE). One 72 kDa protein, was identified by mass spectrometry to be GRP78 (a member of HSP70 protein family) and was up-regulated 2 to 3 fold in infected cells. Up-regulation of GRP78 in DENV-infected cells was confirmed by immuno-staining and confocal microscopy. GRP78 and HSP70 have previously been identified as a component of the DENV receptor complex and blocking of these proteins has been found to inhibit DENV entry into the cell. By confocal microscopy we found that cytoplasmic GRP78 and HSP70 were also up-regulated in DENV-infected cells. The role of cytoplasmic GRP78 and HSP70 in DENV-infected cells has not been established; however there are precedents in other viral infections that cytoplasmic GRP78 and HSP70 could enhance viral protein production. Thus, this thesis shows that (1) the high levels of circulating TNF-α seen in DENV-infection does not influence DENV replication (2) the cellular responses to TNF-α are altered in DENV-infected cells and (3) we have identified two protein chaperones and stress response proteins (GRP78 and HSP70) that are up-regulated during DENV-infection. With the advancement in proteomic techniques since initiation of this project future proteomic analysis could further identify other novel host factors that may either regulate DENV-infection or be involved in a host cell response to DENV-infection and help our understanding of DENV pathogenesis at the protein level.Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 200

Viperin mRNA is induced in DENV-2 infected cells.

<p>Cells were infected with DENV-2 (MOI = 1 or MOI = 3 for MDM) and at various time points pi intracellular RNA was extracted and viperin mRNA and DENV −ve strand RNA quantitated by real time RT-PCR. Results were normalised against control RPLPO mRNA levels and expressed as fold change. Values represent average ± SEM (n = 3). (<b>A</b>) A549; (<b>B</b>) Huh-7; (<b>C</b>) Huh-7.5; (<b>D</b>) MDM. * Significantly different in comparison to 0 h time point, p<0.05.</p

Viperin protein is induced in DENV-2 infected cells.

<p><b>A.</b> Primary MDM were left uninfected, treated with 500 U/ml IFN-α or DENV-2 infected. At 48 h pi cells were lysed and viperin protein analysed by western blot. Blots were re-probed for β-actin and images visualised by chemiluminesence. Images were quantitated using Carestream Molecular Imaging Software and viperin signal normalised against β-actin. <b>B.</b> Primary MDM were DENV-2 (i) or mock (ii) infected and at 24 h pi were fixed and immunostained for viperin and DENV, with detection of stained complexes with anti-rabbit 647 (red) and anti-mouse 488 (green), respectively. Nuclei were stained with Hoechst (blue) and images collected by confocal microscopy. <b>C.</b> Immunolabeling for viperin was quantitated in cells from mock-infected MDM and compared with antigen negative bystander and DENV-2 antigen positive cells of the DENV-2 infected MDM cultures. Values represent average ± SEM. (n = 111 mock; 27 DENV-antigen positive; 136 DENV-antigen negative bystander cells). * = significantly different, p<0.05, Students unpaired t-test. Results of a single experiment are shown which was replicated.</p

Induction of viperin is needed to restrict early viral replication.

<p>Viperin shRNA or control shRNA expressing Huh7 cells were infected with DENV-2 (MOI = 0.1). (<b>A</b>) Supernatant was sampled and infectious virus release quantitated by plaque assay. Values represent average ± SEM (n = 3); At the indicated time point pi cells were lysed, RNA extracted and analysed by real time RT-PCR for (<b>B</b>) viperin mRNA; (<b>C</b>) IFIT1 mRNA. Values represent average ± SEM (n = 4). Results were normalised against control RPLPO mRNA levels and expressed as fold change relative to mock infected cells. * = significant at p<0.05, Students unpaired t-test.</p

Viperin interacts with DENV-2 CA and NS3 protein.

<p>Huh-7 cells were co-transfected with a DENV-2 CA-GFP and viperin-mCherry expression vector (<b>A</b>), or DENV-2 NS3-GFP and expression plasmids for either viperin-mCherry (<b>B</b>), viperin 5′Δ33-mCherry (<b>C</b>) or viperin 3′Δ17-mCherry (<b>D</b>). Slides were analysed on a Zeiss Axioplan microscope and FRET determined by acceptor photobleaching. DIF was calculated from comparison of aligned pre and post-bleach images, from 5–10 regions per cell of at least 10 cells from 2 different experiments. Data are represented as average ± SEM with a significance of p<0.05 for (A), (B) and (C).</p

Viperin is anti-viral against DENV-2 and requires C-terminal regions of the protein.

<p>(<b>A</b>) HeLa cells were transfected with either a viperin-FLAG expression plasmid (<b>i</b>) or a control vector (<b>ii</b>) and at 24 h post transfection infected with DENV-2 (MOI = 1). At 24 h pi cells were fixed and immunolabelled with anti-FLAG (viperin) and anti-dsRNA antibodies with detection of stained complexes with anti-rabbit 647 (red) and anti-mouse 488 (green), respectively. Nuclei were stained with Hoechst (blue) and images collected by confocal microscopy. (<b>B</b>) Huh-7 cells were transfected to express WT viperin or viperin mutants and at 24 h post transfection infected with DENV-2 (MOI = 0.1). 24 h pi RNA was extracted and DENV-2 −ve strand PCR quantitated by real-time RT-PCR. Results were normalised against control RPLPO mRNA levels and expressed as fold change. Values represent average ± SEM (n = 3). * = significantly different to no viperin control, ** = significantly different to no viperin control and WT viperin, p<0.05, Students t-test. Similar experiments to (B) were performed in (<b>C</b>) Huh-7 or (<b>D</b>) A549. Cells were transfected using WT viperin or a 3′Δ17 viperin expression construct and infected as in (B). Supernatant was sampled and analysed for infectious virus release by plaque assay and RNA extracted from infected cells and DENV −ve strand RNA quantitated by real time RT-PCR. Results were normalised against control RPLPO mRNA levels and expressed as fold change relative to 3′Δ17 viperin control. Values represent average ± SEM (n = 3). * = significantly different to WT viperin, p<0.05, Students unpaired t-test.</p

Viperin is anti-viral in primary MDM.

<p>Primary MDM were generated from peripheral blood and transduced with lentiviral particles expressing control td-Tomato or WT viperin. At 24 h post transduction, cells were infected with DENV-2 (MOI = 3). (<b>A</b>) Supernatant was sampled and infectious virus release quantitated by plaque assay. Values represent average ± SEM (n = 3). * p<0.001; (<b>B</b>) Viperin lenti-transduced MDM were DENV-2 or mock infected and at 48 h pi cells were fixed and immunolabelled for viperin and DENV with detection of complexes with Alexa-647 (red) and Alexa-488 (green), respectively. Nuclei were stained with Hoechst (blue) and images collected by confocal microscopy.</p